SOLUTION STRUCTURE OF THE PARAMAGNETIC COMPLEX OF THE N-TERMINAL DOMAIN OF CALMODULIN WITH 2 CE3-1-NMR( IONS BY H)

The solution structure of the dicerium(III) complex of the N-terminal domain of calmodulin (Ce-2-TR1C hereafter) has been solved employing p aramagnetic T-1 relaxation enhancements and pseudocontact shifts intro duced by the Ce3+ ions, together with conventional NOE constraints. Th e use of pseudocontact shift constraints constitutes the first attempt to locate metal ions within a protein structure by NMR. Like calcium( II), paramagnetic cerium(III) has been found to bind to the two metal binding sites of the TR1C fragment of calmodulin in a cooperative mann er. Due to the presence of pseudocontact interactions between the Ce3 ions and protons of the 76-residue protein, the H-1 NMR spectra of th e complex show resonances shifted between +22 and -9 ppm. Eighty perce nt of its proton resonances could be assigned through a standard appro ach using TOCSY/COSY and NOESY spectra and through 1D NOE difference s pectra for the broad resonances of protons close to the paramagnetic i ons. A family of structures was calculated by means of the torsion ang le dynamics program DYANA [Guntert, P., Mumenthaler, C., & Wuthrich, K . (1996) XVIIth International Conference on Magnetic Resonance in Biol ogical Systems (Abstract)] using 1012 NOEs. Longitudinal proton relaxa tion times helped to roughly define the position of the metal ions wit hin the protein. A total of 381 pseudocontact shift constraints, whose evaluation and use are critically discussed, have then been added to further refine the metal coordinates within the protein frame and to i mprove the structure resolution, A dramatic resolution improvement of the metal coordinates together with a sizable resolution improvement i n the regions close to the paramagnetic centers, where the number of N OEs is low, is observed. The good quality of the solution structure pe rmitted a meaningful comparison with the solid-state structure of calc ium-loaded calmodulin at 1.7 Angstrom resolution [Chattopadhyaya, R., Meador, W, E., Means, A. R,, & Quiocho, F. A, (1992) J. Mel. Biol. 228 , 1177]. The Ce-2-TR1C complex is overall more compact than the Ca for m.